Recombinant Solanum lycopersicum Serine/threonine-protein phosphatase 5 (PP5)

What are the known isoforms of tomato PP5 and how do they differ?

Two main isoforms of tomato PP5 have been characterized:

• 62 kDa isoform (NP_001300937.1)

• 55 kDa isoform (NP_001234232.2)

These isoforms result from alternative splicing mechanisms, which significantly affect their subcellular localization and potentially their function . The subcellular targeting of these isoforms is determined by specific sequence elements generated through this alternative splicing process, as demonstrated in research by de la Fuente van Bentem et al. (2003). This differential localization likely reflects distinct roles of each isoform in cellular compartment-specific signaling pathways.

How conserved is PP5 across plant species compared to tomato?

PP5 is highly conserved across plant species, though with important variations. Comparisons between tomato PP5 and homologs in other plants reveal:

This conservation suggests fundamental roles for PP5 in plant signaling pathways, while species-specific variations may reflect adaptations to different environmental conditions and stressors .

What expression systems are most effective for producing recombinant tomato PP5?

The effectiveness of expression systems for recombinant tomato PP5 production varies based on research objectives:

For standard biochemical characterization, E. coli expression using vectors such as pcDNA3.1-C-(k)DYK has been successful for PP5 production . Purification protocols typically involve affinity chromatography using the C-terminal tag, followed by size exclusion chromatography to separate the different oligomeric states that may form.

How can researchers assess the catalytic activity of recombinant tomato PP5?

A methodological approach to assess tomato PP5 catalytic activity includes:

• Substrate selection: Common substrates include:

  • p-nitrophenyl phosphate (pNPP) - artificial colorimetric substrate

  • Phosphorylated peptides corresponding to known PP5 substrates

  • 32P-labeled proteins isolated from plant tissues

• Reaction conditions optimization:

  • Buffer: Typically Tris-HCl (pH 7.0-8.0) with MnCl₂ or MgCl₂

  • Temperature: Usually 30°C for plant phosphatases

  • Incubation time: 10-30 minutes, depending on enzyme concentration

• Activity measurement:

  • For pNPP: Measure absorbance at 405 nm

  • For phosphopeptides: Use malachite green assay for released phosphate

  • For 32P-labeled substrates: Measure released 32P by scintillation counting

• Inhibitor controls:

  • Include okadaic acid (10-100 nM) as PP5 is sensitive to this inhibitor

  • Compare with other phosphatase inhibitors (microcystins, calyculin A) to confirm specificity

The specific activity should be expressed as nmol phosphate released per minute per mg of enzyme under standard conditions.

What strategies help overcome stability issues with recombinant tomato PP5?

Tomato PP5 can present stability challenges during recombinant expression and purification. Effective strategies include:

• Expression optimization:

  • Lowering induction temperature (16-18°C)

  • Co-expression with molecular chaperones (GroEL/GroES)

  • Using strains optimized for disulfide bond formation (e.g., Origami)

• Buffer optimization:

  • Include 10-15% glycerol as a stabilizing agent

  • Add reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Test different pH ranges (pH 7.0-8.5)

  • Include divalent cations (Mn²⁺, Mg²⁺) at 1-5 mM

• Storage considerations:

  • Flash freeze in liquid nitrogen with 20% glycerol

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles

  • For short-term storage, keep at 4°C with protease inhibitors

• Heat stability assessment:

  • Similar to studies with other recombinant tomato proteins, thermal stability can be evaluated by heating at various temperatures (50-99°C) for different durations

  • Analysis by SDS-PAGE and activity assays post-heating provides insights into thermal tolerance

How does tomato PP5 interact with heat shock protein 90 (Hsp90) complexes?

Tomato PP5 interacts with Hsp90 through its N-terminal tetratricopeptide repeat (TPR) domain. The methodology to study this interaction involves:

• Co-immunoprecipitation assays:

  • Use anti-PP5 antibodies to pull down protein complexes from tomato extracts

  • Analyze precipitated proteins by Western blotting with anti-Hsp90 antibodies

  • Reverse co-IP using anti-Hsp90 antibodies to confirm interaction

• TPR domain mutation studies:

  • Generate PP5 constructs with mutations in key TPR residues

  • Express wildtype and mutant proteins in plant expression systems

  • Compare Hsp90 binding efficiency using pull-down assays

• Functional consequences assessment:

  • TPR "dominant-negative" experiments reveal that overexpression of just the TPR domain can disrupt native PP5-Hsp90 interactions

  • This disruption affects numerous signaling pathways including hormone responses

Research indicates that PP5 appears to play a regulatory role in Hsp90 chaperone activity, potentially modulating client protein maturation and stabilization. PP5 has been observed in Hsp90 heterocomplexes containing other proteins, including heme-regulated eIF2α kinase and heat shock factor 1 (Hsf-1), where it functions as a negative modulator .

What is the role of tomato PP5 in drought stress responses?

Tomato PP5 appears to function within a complex network of stress signaling pathways involved in drought responses. A methodological framework to study this includes:

• Expression analysis during drought stress:

  • qRT-PCR analysis of PP5 transcript levels at different drought timepoints

  • Western blot analysis of PP5 protein levels

  • Comparison with known drought-responsive genes (e.g., AREB/ABF family)

• Phosphatase activity measurements during stress:

  • Extract native PP5 from drought-stressed and control plants

  • Measure activity using specific substrates

  • Compare with other phosphatases (e.g., PP2C) known to be involved in drought responses

• Transgenic approaches:

  • Generate PP5-overexpressing and PP5-silenced tomato plants

  • Subject to controlled drought conditions and assess:

    • Physiological parameters (water loss, stomatal conductance)

    • ABA-related signaling components

    • Transcriptome changes

While direct evidence for PP5 in tomato drought resistance is still emerging, research in related pathways shows that protein phosphatases play critical roles in ABA signaling and stress adaptation. For instance, PP2C phosphatases in tomato show elevated transcript levels during drought stress, with specific members (SlPP2C22, SlPP2C30, and SlPP2C52) being particularly responsive .

How does PP5 contribute to hormone signaling networks in tomato?

PP5 intersects with multiple hormone signaling pathways in tomato. A structured research approach includes:

• Hormone-responsive expression analysis:

  • Treat tomato plants/cells with different hormones (ABA, auxin, ethylene, gibberellins)

  • Monitor PP5 expression changes by qRT-PCR and Western blotting

  • Identify hormone-responsive elements in the PP5 promoter region

• Protein-protein interaction studies:

  • Use yeast two-hybrid or BiFC assays to identify interactions with hormone signaling components

  • Confirm in planta using co-immunoprecipitation

  • Map interaction domains using truncated protein versions

• Phosphorylation site identification:

  • Use mass spectrometry to identify hormone-regulated phosphorylation sites on PP5

  • Generate phospho-mimetic and phospho-null mutants

  • Assess impact on PP5 activity and interactions

Research indicates that PP5 interacts with hormone receptors and signaling components. For example, PP5 has been shown to affect the nuclear translocation of hormone receptors via its association with Hsp90, and the overexpression of the PP5 TPR domain facilitates the dissociation of peroxisome proliferator-activated receptors (PPARα and PPARβ) from Hsp90 .

How can researchers distinguish between PP5 and other phosphatases in functional studies?

Distinguishing the specific functions of tomato PP5 from other phosphatases requires a multi-faceted approach:

• Inhibitor profiling:

  • PP5 is sensitive to okadaic acid, microcystins, nodularin, calyculin A, tautomycin and cantharidin

  • Create inhibition profiles using varying concentrations of these inhibitors

  • Compare IC₅₀ values with those for PP1, PP2A, and other phosphatases

• Substrate specificity determination:

  • Test phosphopeptide libraries with varied sequences

  • Identify preferential recognition motifs for PP5

  • Compare with substrate preferences of other phosphatases

• Genetic approaches:

  • Use CRISPR-Cas9 to generate PP5 knockout lines

  • Complement with mutant versions or other phosphatases

  • Assess phenotypic rescue to determine unique functions

• Interaction network mapping:

  • Use proximity labeling techniques (BioID, APEX) with PP5 as bait

  • Compare interactome with other phosphatases

  • Identify unique PP5 partners to infer specific functions

It's crucial to note that studies using phosphatase inhibitors often attribute effects to PP1 or PP2A without considering PP5's potential involvement, as PP5 is also sensitive to these common inhibitors .

What considerations are important when investigating PP5 roles in tomato stress signaling pathways?

When designing experiments to investigate tomato PP5 in stress signaling:

• Stress type and intensity standardization:

  • Define clear stress parameters (e.g., soil water content for drought)

  • Use time-course sampling to capture dynamic responses

  • Include multiple stress intensities to identify threshold effects

• Multi-level analysis integration:

  • Combine transcriptomics, proteomics, and phosphoproteomics

  • Map phosphorylation/dephosphorylation events temporally

  • Correlate PP5 activity with specific substrate modifications

• Consideration of genetic background effects:

  • Compare responses in different tomato varieties/accessions

  • Include wild relatives (e.g., S. pennellii) known for stress tolerance

  • Use introgression lines to map genetic interactions

• Methodology for separating overlapping stress responses:

  • Design factorial experiments with multiple stress types

  • Use statistical approaches to identify stress-specific vs. general responses

  • Consider hormone crosstalk (particularly ABA and ethylene)

Studies in tomato have revealed that wild relatives possess enhanced drought tolerance mechanisms compared to cultivated varieties, with specific ABA-responsive genes showing differential expression patterns between species (e.g., S. pennellii vs. S. lycopersicum) . These genetic resources provide valuable tools for dissecting PP5's role in stress adaptation.

What techniques can resolve contradictory data about PP5 functions in different experimental systems?

Resolving contradictory findings about PP5 functions requires systematic troubleshooting:

• System-specific variables identification:

  • Compare protein extraction methods and activity preservation

  • Assess post-translational modifications across systems

  • Examine expression levels relative to endogenous PP5

• Isoform-specific analysis:

  • Design isoform-specific primers/antibodies

  • Create constructs expressing single isoforms

  • Analyze subcellular localization patterns

• Context-dependent activation assessment:

  • Test activity under varied ion concentrations

  • Examine effects of cellular stressors on activity

  • Investigate protein-protein interactions that may modulate function

• Reconciliation framework:

  • Map contradictory results to specific experimental conditions

  • Test unified hypotheses that account for divergent observations

  • Develop in vitro systems that recapitulate in vivo complexity

For example, contradictory results regarding PP5's role in p53 signaling have been clarified by recognizing that while PP5 can dephosphorylate p53 in vitro, in vivo it likely acts upstream in the pathway, potentially by augmenting actions of a glucocorticoid receptor-induced kinase such as serum-glucocorticoid inducible kinase-1 (SGK-1) .

How can modern genomic approaches advance understanding of tomato PP5 evolution and function?

Advanced genomic approaches offer new opportunities for PP5 research:

• Comparative genomics methodology:

  • Analyze PP5 orthologs across Solanaceae family members

  • Identify conserved regulatory elements in promoter regions

  • Map selection pressure on different protein domains

• GWAS and QTL mapping integration:

  • Use tomato diversity panels for association mapping

  • Correlate PP5 sequence/expression variation with phenotypic traits

  • Leverage tomato introgression lines and MAGIC populations

• Epigenetic regulation investigation:

  • Profile DNA methylation at the PP5 locus under various conditions

  • Analyze histone modifications associated with expression changes

  • Investigate potential small RNA-mediated regulation

• Single-cell approaches application:

  • Map PP5 expression at cellular resolution during development

  • Identify cell type-specific roles in stress responses

  • Examine phosphorylation dynamics in specific cell populations

The available tomato genomic resources, including high-quality genome sequences, transcriptomic data, and populations like the ToMAGIC multi-parent advanced generation inter-cross, provide powerful tools for such investigations . These resources facilitate fine mapping of traits and identification of candidate genes that may interact with PP5 in various physiological processes.

What emerging technologies might advance tomato PP5 research?

Several cutting-edge technologies hold promise for tomato PP5 research:

• Proximity-dependent labeling techniques:

  • BioID or TurboID fusion with PP5 to identify transient interactors

  • APEX2 for ultrastructural localization and temporal interaction mapping

  • Split-BioID for conditional interaction studies

• Real-time phosphatase activity sensors:

  • Develop FRET-based sensors for dynamic PP5 activity measurement

  • Create genetically encoded reporters for specific PP5 substrates

  • Implement optogenetic control of PP5 activity

• Cryo-EM for structural biology:

  • Resolve full-length PP5 structure with regulatory domains

  • Capture PP5-substrate complexes at different stages

  • Visualize PP5 within larger protein complexes (e.g., with Hsp90)

• Synthetic biology approaches:

  • Design orthogonal PP5 variants with engineered substrate specificity

  • Create chemical-genetic systems for rapid PP5 inhibition

  • Develop PP5-based biosensors for stress conditions

These approaches could provide unprecedented insights into PP5 function, regulation, and its role in tomato stress adaptation, potentially leading to applications in improving crop resilience.